The present invention is directed to pharmaceutical compositions, and primarily to topically applied ophthalmic compositions comprising as the active ingredient one or more compounds having the ability to chelate calcium ions, e.g. intracellular calcium ions. The pharmaceutical compositions are useful for reducing intraocular pressure in animals of the mammalian species. In another aspect, the present invention is directed to administering such formulations and compositions to animals of the mammalian species (including humans) for reducing intraocular pressure in the eye. Yet another aspect of the invention concerns methods of delivering a drug to a cell by joining the drug to an acetylcholine or psuedoacetylcholine group, thereby targeting cells containing cell surface acetylcholinesterase or acetylcholine receptors. The drug preferably contains carboxylic acid groups that are neutralized using alkyl ester blocking groups to form a prodrug. Following cell surface binding, the prodrug is able to pass through the cell membrane whereupon the neutralizing blocking groups are removed through the action of intracellular enzymes to release the drug. Preferred prodrugs include acetoxymethyl esters of BAPTA.
Glaucoma is an optical neuropathy associated with elevated intraocular pressures which are too high for normal function of the eye, and results in irreversible loss of visual function. It is estimated in medical science that glaucoma afflicts approximately 2 percent of the population over the age of forty years, and is therefore a serious health problem. Ocular hypertension, i.e. the condition of elevated intraocular pressure, which has not yet caused irreversible damage, is believed to represent the earliest phase of glaucoma. Many therapeutic agents have been devised and discovered in the prior art for the treatment or amelioration of glaucoma and of the condition of increased intraocular pressure which precedes glaucoma. Other compounds known to be useful in treating intraocular pressure are disclosed in the following patents.
U.S. Pat. No. 3,467,756 describes anti-glaucoma and intraocular hypotensive compositions which contain in an ophthalmic vehicle 10,11-dihydro-5-(3-methylaminopropyl)-5,10-epoxy-11-hydroxy-5H-dibenzo [a,d]cycloheptene or related derivatives.
U.S. Pat. No. 4,197,301 describes ophthalmic compositions which contain 1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-furanylcarbonyl)piperazine, also known under the name xe2x80x9cprazosinxe2x80x9d.
U.S. Pat. No. 4,565,821 describes a method of topically administering certain dopamine antagonists to reduce ocular hypertension and to treat glaucoma.
U.S. Pat. No. 4,886,815 describes a method for treating retinal edema by administration of dopaminergic antagonists to a patient suffering from such conditions.
U.S. Pat. No. 5,066,664 describes 2-hydroxy-2-alkylphenylamino)-oxazolines and thiazolines as anti-glaucoma and vasoconstrictive agents.
U.S. Pat. No. 5,091,528 describe 6 or 7-(2-imino-2-imidazolidine)-1,4-benzoxazines as adrenergic agents useful for treating glaucoma.
The foregoing and other anti-glaucoma and ocular hypotensive compounds and agents of the prior art do not provide such treatment or cure for glaucoma and ocular hypertension which is satisfactory in all respects. Therefore, the pharmacological and related arts and sciences continue searching for additional and better anti-glaucoma and ocular hypotensive agents.
1,2-bis(2-aminophenoxy)ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (BAPTA) is a specific Ca2+ chelator that has been used to clamp extracellular Ca2+ to desired levels. On the other hand, the acetoxymethyl ester of BAPTA (BAPTA-AM) the uncharged esterified form of the parent compound is used to clamp intracellular Ca2+(Ca2+i). BAPTA-AM penetrates biological cell membranes and is hydrolyzed by intracellular esterases yielding the original charged impermeable form of the compound once again capable of buffering/clamping Ca2+. (This is reported in Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals; Richard P. Haugland 1992-1994; Section 20: Calcium Indicators, Chelators and Ionophores, pages 119-128.)
Surprisingly it has been discovered in accordance with the present invention that calcium chelating agents are effective as anti-glaucoma agents and as agents for reducing intraocular pressure, when such agents are applied to the mammalian eye in a pharmaceutical composition, preferably in a topical ophthalmic composition. Accordingly, in one aspect the present invention relates to a method of treating glaucoma, or ocular hypertension by topically administering to the mammalian eye an ophthalmic composition which contain an effective amount of a calcium chelating agent. A preferred example of calcium chelating agents suitable as the active ingredients of the ophthalmic compositions of the invention are: 
and esters, e.g. the lower alkyl and alkoxyalkyl esters thereof. Such esters may be represented by the general formula 
wherein R is lower alkyl, e.g. an alkyl radical having from 1 to 6 carbon atoms, or xe2x80x94R1xe2x80x94Oxe2x80x94Cxe2x80x94R2 wherein R1 is a lower alkylene radical, e.g. an alkylene radical having from 1 to 6 carbon atoms, and R2 is R, as defined above. R, R1 and R2 may be interrupted with O or N radicals as in alkyloxy alkyl and alkylaminoalkyl moieties, e.g. R1 may be xe2x80x94CH2xe2x80x94N(CH3)CH2CH2xe2x80x94 and xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94. Preferably, R1 and R2 will comprise from 1 to 4 carbon atoms, e.g. 1 carbon atom.
While not wishing to be bound by theory it is believed that calcium chelating agents, e.g. 1,2-bis (2-aminophenoxy) ethane-N, N, Nxe2x80x2, Nxe2x80x2-tetraacetic acid (BAPTA) or the acetoxymethyl ester of BAPTA (BAPTA-AM), are useful for treating hypertensive glaucoma, because intracellular (Ca2+i) is fundamental in the activation/control and modulation of epithelial fluid secretion. Thus, clamping/lowering the level of Ca2+i in the ciliary epithelium, the tissue responsible for aqueous humor formation and a determinant of intraocular pressure (IOP), will reduce inflow and therefore IOP.
The ophthalmic compositions of the invention contain the active ingredient in a concentration range of approximately 0.0001 to 0.1 percent weight by volume. The composition itself includes, in addition to the active ingredient, such excipients which are per se well know in the art for preparing ophthalmic compositions, particularly ophthalmic solutions. In accordance with a method of the invention the ophthalmic compositions, preferably ophthalmic solutions, are applied topically to the mammalian eye approximately 1 or 2 times daily.
In another aspect, the present invention also relates to methods and compositions for delivering drugs to cells having acetylcholinesterase or acetylcholine receptors on their cell surfaces. The drugs preferably contain at least one carboxylic acid group, and are formulated as a prodrug, with the carboxylic acid group(s) neutralized by derivatization with an alkyl ester blocking group. Such prodrugs also comprise a di- ) or trimethyl ammonium moiety, an isobutyl or t-butyl moiety, or a sterically similar structure and are able to bind to acetylcholinesterase or an acetylcholine receptor.
As stated above, the prodrugs are designed to be liberated as a charged species once within the target cell through the action of intracellular esterases. While this aspect of the invention applies to any carboxylic acid-containing drug to be delivered to the cytoplasm of cells having cell surface acetylcholine-binding proteins, in a preferred example the prodrug comprises a calcium chelator compound as described above, together with at least one hydrophobic blocking group such as the acetoxymethyl ester (AM) blocking groups mentioned above. In a particularly preferred example, the calcium chelator prodrug comprises the non-polar BAPTA derivative BAPTA-AM; once inside the cell, esterases convert the uncharged BAPTA-AM to the polar calcium chelator BAPTA. The conversion of the prodrug to a polar form limits the tendency of neutral compounds to quickly leak out of the cell.
The method of this aspect of the invention involves providing the prodrug to cells such as neural cells (for example, ciliary epithelial and retinal cells) having cell surface acetylcholinesterase or acetylcholine receptors by joining the prodrug to a chemical structure mimicking the acetylcholine molecule. Hasan et al., J. Biol. Chem. 256:7781-7785 (1981), Hasan and Cohen, J. Biol. Chem. 255:3898-3904 (1980) and Cohen et al., J. Biol. Chem. 257:14087-14092 (1982), each of which is incorporated by reference herein as part of this specification, have shown in structural studies of inhibitors of acetylcholinesterase that a substantial determinant of acetylcholinesterase-substrate binding specificity is the trimethyl ammonium structure of acetylcholine. Analogs of acetylcholine that have groups sterically resembling this trimethyl ammonium structure, such as, without limitation, a dimethyl ammonium or t-butyl moiety, are also able to bind and activate acetylcholinesterase, demonstrating the relative importance of the tertiary structure of the acetylcholinesterase substrate over the positive charge of the acetylcholine.
Thus, the prodrugs of this aspect of the invention are joined to an acetylcholine ester or pseudo-acetylcholine ester (collectively referred to herein as a xe2x80x9cpACExe2x80x9d moiety) that is able to bind acetylcholinesterase or an acetylcholine receptor; the pACE moiety may then be liberated from the prodrug by acetylcholinesterase-mediated hydrolysis, or the entire prodrug-pACE molecule may be transported through the plasma membrane. In either case the drug is then liberated within the cell through the action of cytoplasmic esterases.